Practical Synthesis of Antimicrobial Long Linear Polyamine Succinamides

There are many severe bacterial infections notorious for their ability to become resistant to clinically relevant antibiotics. Indeed, antibiotic resistance is a growing threat to human health, further exacerbated by the lack of new antibiotics. We now describe the practical synthesis of a series of substituted long linear polyamines that produce rapid antibacterial activity against both Gram-positive and Gram-negative bacteria, including meticillin-resistant Staphylococcus aureus. These compounds also reduce biofilm formation in Pseudomonas aeruginosa. The most potent analogues are thermine, spermine, and 1,12-diaminododecane homo- and heterodimeric polyamine succinic acid amides. They are of the order of activity of the aminoglycoside antibiotics kanamycin and tobramycin as positive controls. Their low human cell toxicity is demonstrated in ex vivo hemolytic assays where they did not produce even 5% hemolysis of human erythrocytes. These long, linear polyamines are a new class of broad-spectrum antibacterials active against drug-resistant pathogens.


■ INTRODUCTION
Antimicrobial resistance (AMR) is one of the top 10 current global health threats. 1,2 The continuous increase of AMR among major bacterial pathogens is expected to result in 10 million deaths per year by 2050 with an estimated US$1 trillion in health-care costs globally. 3,4 Estimates have indicated that up to US$100 trillion could be lost to the global economy due to decreased productivity. 3 Despite the urgent need for investments in research and development of antibiotics, only two new antibiotic classes (oxazolidinones and lipopeptides) have emerged and been approved for clinical use. 5 As a response to the lack of antibiotics in the clinical pipeline and the growing spread of antibiotic resistance, the WHO have generated a priority list to inform and direct research to tackle specific antibiotic-resistant human bacterial pathogens through the development of new and effective antibiotics. 6 Accordingly, we have examined the activity of novel linear polyamines against seven major human bacterial pathogens identified on this list (Enterococcus faecalis, E. faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Escherichia coli) and one emerging pathogen (S. epidermidis).
The positive charges of the amino groups along these linear polyamines 7 and the elongated polyamine amides are potentially a key factor in their rapid antimicrobial activity against both Gram-positive and Gram-negative bacteria including meticillinresistant S. aureus (MRSA), potentially primarily acting by depolarization of the cytoplasmic membrane and permeabilization of the bacterial outer membrane. 8,9 Linking two of the same or different polyamines via amide bonds can be achieved by introducing a carboxylic acid group on the first polyamine, then coupling to a free primary amine in the second polyamine. If the addition of positive charges 10−12 increases the antimicrobial activity of linear polyamines, 13−17 synthesizing homo-or heterodimeric polyamines will increase the total net charge compared to their monomeric counterparts. 18

3) 3 and Selective Protection of the Reactive Amines on Polyamines
A two-step procedure was used to form tetraamine 3 which incorporates the N 1 ,N 12 -bis(3-aminopropyl) pattern of substituents on 1,12-diaminododecane. 19,20 Using the trivial nomenclature for linear polyamines of the (poly)methylene count, where tetraamine thermine 4 is indicated as 3.3.3 and spermine 5 is known as 3.4.3, this long-chain target tetraamine is accurately described as 3.12.3. Commercially available 1,12diaminododecane 1 was reacted with two equivalents of acrylonitrile in EtOH at 20°C to undergo two 1,4-Michael addition reactions to obtain dinitrile 2 20 in 87% yield (see Figures S1 and S2). The formation of some tri-Michael adducts was observed. 13 C NMR spectroscopy showed a nitrile carbon signal of low intensity at 118.8 ppm. Using LiAlH 4 in anhydrous THF was investigated to reduce the nitrile functional groups to primary amines, but this gave a complex mixture. 21 Pd/C (10%) was investigated as a catalyst under hydrogen gas with little success, 22 likewise using Raney nickel as a catalyst. 23 However, the nitrile functional groups were successfully reduced to primary amines using catalytic Raney nickel and sodium hydroxide (co-catalyst) under a hydrogen pressure of 1 bar to afford tetraamine 3 in 75% yield (see Figure S3). 24,25 The ability to covalently link polyamines requires selective protection, in order to avoid unwanted side products. However, the protection of three amines out of four is always low yielding and needs substantial chromatographic purification. Monoprotection using benzyl chloroformate (CbzCl) or di-tertbutyldicarbonate (Boc anhydride) is not practical due to low yields and requires more substantial chromatographic purification. Geall and Blagbrough reported that using trifluoroacetyl as a protecting group for one amine out of four can be controlled by decreasing the temperature and the concentration. 26 Subsequent removal under basic conditions makes trifluoroacetyl an ideal protecting group compared to Cbz and Boc for the purpose of gram-scale protection of polyamines in preparing unsymmetrical polyamine amides. The ratio between −NH 2 and ethyl trifluoroacetate (the source of the protecting group) is important not only to avoid protection of both primary amines but also to avoid protection of the more sterically hindered secondary amines on 3, 4, and 5. Thus, using this approach, via mono-trifluoroacetamides 6, 7, and 8, protection of the three remaining amino groups in the fully protected derivatives 9, 10, and 11, respectively, the corresponding three tri-Boc protected derivatives 12, 13, and 14 were obtained following trifluoroacetyl removal, Figure 1.

Synthesis of Polyamine Amide-Succinic Half Acids and Their Conversion into Homo-and Heterodimeric Linear Polyamine Succinamides
Succinic anhydride was selected to link two of the same or different linear polyamines, introducing a short (4 carbon) spacer and a useful carboxylic acid functional group from which amides may be obtained, for example, from the same or different long linear polyamines incorporating a N 1 ,N 12 -bis(3-aminopropyl)-1,12-diaminododecane moiety. 27 Compounds 15,16, and 17 were synthesized by the addition of one equivalent of succinic anhydride to a solution of each tri-Boc protected polyamine 12, 13, and 14 in anhydrous pyridine at 20°C, Figure  1. All spectral data confirmed that the reactions successfully occurred.
The 1 H-13 C HMBC NMR spectrum of succinic amide acid 17, the tri-Boc compound derived from spermine (3.4.3, 5) shows that there are two triplets at 2.45 and 2.58 ppm in the 1 H NMR spectrum ( Figure S5), which represent the two CH 2 groups between the amide group and the carboxylic acid, and carbon signals at 173.4 and 174.8 ppm in the 13 C NMR spectrum, which represent the carbonyl carbons of the carboxylic acid and the amide group. 1 H-13 C cross-peaks in the HMBC NMR spectra between the two triplets and the two carbon signals were observed, see Figure S4. Compounds 15,16, and 17 were separately coupled to one equivalent of the primary amine 12 to obtain the corresponding target homo-and heterodimeric protected polyamines 18, 19, and 20 in good yield by the addition of one equivalent of HBTu to activate the carboxylic acid in anhydrous DMF at 20°C. 1 H NMR spectra show a singlet at 2.52 ppm integrating for the four protons between the two new amide groups (RNHCOCH 2 CH 2 CONHR) rather than a triplet, as observed for succinic amide half acids 15, 16, and 17, Figure S5. Also, there is one carbonyl signal observed at ∼175 ppm with higher intensity, from both the amide carbonyl groups, and the disappearance of the carboxylic acid signal in the 13  The antibacterial activity was measured for compounds 21, 22, and 23 on different bacterial strains (Table 1). In particular, activity was found against P. aeruginosa PAO1 and S. aureus SH1000, at levels comparable to those of the positive controls, kanamycin (MIC = 32 μg/mL) and tobramycin (MIC = 1−2 μg/mL). In order to investigate if there was any significant difference caused by the counterions, homo-dimer 21 was prepared not only as its poly-(hexa)-TFA salt but also as its poly-HCl salt, by Boc-protecting group removal with 4 M HCl in 1,4dioxane at 20°C. The solution was stirred for 18 h, then concentrated in vacuo, and lyophilized to yield the desired product as a white powder. The poly-HCl salt of homo-dimer 21 was as equally potent as its hexa-TFA salt against S. aureus SH1000, MIC = 4 μg/mL. Also, there were good antibiofilm data for all three polyamine succinamides, with 8−32 μg/mL MIBC antibiofilm levels against PAO1 and 4−64 μg/mL MIBC against SH1000 (Table 2). For toxicity quantified by measurement on human erythrocytes (hRBCs), even at concentrations of 512 μg/mL, there was less than 5% hemolysis of hRBCs, shown in the hemolysis HC5 concentration data for compounds 21, 22, and 23 (Table 3) where hemolysis HC5 is the concentration required to induce hemolysis of 5% of the erythrocyte population.
Quaternary ammonium compounds (QACs) typically contain trimethylammonium or benzyldimethyl alkylammo-nium units where, as they are quaternized amines, the positive charge is permanent, typically with a corresponding chloride or bromide counterion. The three target long linear polyamines reported here are not QACs. They are neither quaternary nor permanently positively charged. QACs are widely applied as biocides in household and in numerous industrial products used for, for example, water treatment, antifungal treatment in horticulture, in pharmaceutical and everyday consumer products as preservatives, foam boosters, and detergents. Therefore, QACs occur in the aquatic and terrestrial environment all over the world. The excessive use of QACs has resulted in the emergence of antibiotic-resistant bacteria as QACs act as detergents targeting the lipid wall. Therefore, they do kill microbes working against a broad range of microorganisms, but still their modes of action are not finalized. 28 As Hegstad and coworkers reviewed, 29 the resistance toward QACs is widespread among a diverse range of microorganisms. This resistance occurs by several mechanisms, for example, modifications in the composition of the membrane, expression of efflux pump genes, and/or expression of stress response and repair systems. Development of resistance in both pathogenic and nonpathogenic bacteria has been related to application in human medicine and the food industry. QACs in cosmetic products inevitably come into intimate contact with the skin or mucosal linings in the mouth and thus they are likely to add to the selection pressure toward more QAC-resistant microorganisms among the skin or mouth flora. There is increasing evidence of co-resistance and cross-resistance between QACs and a range of other clinically important antibiotics and disinfectants. The use of QACs may have driven the fixation and spread of certain resistance cassette collectors (class 1 integrons), currently responsible for a major part of AMR in Gram-negative bacteria. More indiscriminate use of QACs such as in cosmetic products may drive the selection of further new genetic elements that will aid in the persistence and spread of AMR and thus in further limiting our few remaining treatment options for microbial infections. 29 Woster and co-workers have reported 9 a series of substituted diamines that produce rapid bactericidal activity against both Gram-positive and Gram-negative bacteria. The mode of action of these antibacterial diamines is by targeting bacterial membranes. The linear diamines are based on a dithioacylated 3.5.3 polyamine skeleton, terminating in lipophilic phenyl thioureas. They also reduce biofilm formation and promote biofilm dispersal in P. aeruginosa. These exciting new compounds typically display MIC of 2 μg/mL (against MRSA) and 8 μg/mL (against P. aeruginosa). These linear diamines act primarily by rapid depolarization of the cytoplasmic membrane and permeabilization of the bacterial outer membrane. Also significant are the results in human cell toxicity assays, where they showed limited adverse effects leading Woster and colleagues to conclude that such linear polyamine-derived diamines represent a new class of broadspectrum antibacterials against drug-resistant pathogens. 9 Haldar and co-workers reported 30 di-Phe-derived lipophilic triamides of norspermidine (3.3) are membrane-active with excellent selective antibacterial activity against various wild-type bacteria (Gram-positive and Gram-negative) and drug-resistant bacteria. These are diamines from the two aromatic amino acids (Phe). Such rapidly bactericidal natural and synthetic membrane-active antibacterial agents offer hope as potential solutions to the problem of bacterial resistance as their membrane-active nature imparts a low propensity for the development of resistance and they have potential as therapeutic agents to tackle multidrug-resistant bacterial infections. In related studies, Konai and Haldar reported 31 membrane-active lipophilic norspermidine-derived triamides that are di-Lys conjugates and therefore they typically carry four positive charges. As we have shown herein, the toxicity evaluated against the lysis of hRBCs showed that such linear tetraamines did not cause significant hemolysis. To demonstrate that they are effective as selective antibacterial agents, the hemolytic activity assay for the common antiseptics, benzalkonium chloride (C12 from a range of C8−C18, BAC-12) and didecyl dimethyl ammonium bromide (DDAB-10), and other cationic amphiphiles, QACs such as 1-dodecyl trimethyl ammonium bromide (DTAB) and 1-hexadecyl trimethyl ammonium bromide (C16 is cetyl, CTAB), were also performed. The QACs are highly toxic, resulting in hRBC lysis at very low concentrations (27− 218 μg/mL), 31 whereas our compounds are antibacterial at very  low concentrations and remain nontoxic even at significantly higher concentrations of >512 μg/mL. Such compounds are selective antibacterial agents as they do not display toxicity against mammalian HeLa cells, determined using the MTT assay and found to be >70 μg/mL which is manyfold higher compared to the concentration required for bactericidal activity. 31 ■ CONCLUSIONS The practical synthesis of three long linear polyamine amides that produce rapid antibacterial activity against both Grampositive and Gram-negative bacteria, including MRSA, is described. All these compounds also effectively reduce biofilm formation in P. aeruginosa. These potent analogues are thermine, spermine, and bis-N 1 ,N 12 (3-aminopropyl)-1,12-diaminododecane homo-and heterodimeric polyamine succinic acid amides with activity of the order of the aminoglycoside antibiotics kanamycin and tobramycin as positive controls. These data are important because they reveal that long, linear polyamines are a new chemical class of broad-spectrum antibacterial agents active against drug-resistant pathogens. Such polyamines cause a low (<5%) incidence of hRBC hemolytic toxicity. These data show and confirm that some linear polyamine analogues possess antibacterial activity against both Gram-positive and Gramnegative bacterial strains. Column chromatography was performed over silica gel 60−120 mesh (purchased from Sigma-Aldrich, UK) using different ratios of MeOH, EtOH, ethyl acetate, DCM, and aqueous ammonia (32%) as eluents. Thin-layer chromatography (TLC) was performed over silica gel using aluminum-backed sheets coated with Kieselgel 60 F 254 purchased from Merck (UK). Ninhydrin TLC spray reagent was used for detecting amine functional groups [ninhydrin (0.2 g) in 100 mL EtOH].

Bacterial Strains, Culture Conditions, Minimum Inhibitory Concentration Determination
Bacterial strains used in this study are listed in Table 1. S. aureus, S. epidermidis, E. faecalis, and E. faecium were grown on tryptic soy agar (TSA; Sigma-Aldrich, UK) for 18 h at 37°C. K. pneumoniae, A. baumannii, P. aeruginosa, and E. coli were grown on Luria−Bertani agar (LBA; Sigma-Aldrich, UK) for 18 h at 37°C. The minimum inhibitory concentration of polyamine compounds against the above bacterial species was determined using the broth microdilution method as described by the Clinical and Laboratory Standards Institute (CLSI). 32 Individual pure colonies of the above bacterial species were used to inoculate separate 15 mL polystyrene test tubes (Thermo Fisher) containing 3 mL of cation-adjusted Mueller−Hinton broth (MHB; Oxoid). Growth agar and broth were made, according to manufacturer's instructions. Bacterial cultures were incubated for 18 h at 37°C with shaking at 180 rpm (New Brunswick Innova 44/R incubator). The 18 h bacterial cultures were subsequently diluted 1:100 in fresh MHB and cultured at 37°C with shaking at 180 rpm to an exponential phase of growth, defined as reaching an absorbance (OD 600nm ) within the range of 0.5−0.6. Absorbance was measured using a 1 mm cuvette and DS-11 spectrophotometer (DeNovix). Polyamine compounds were reconstituted in sterile deionized water. Compounds were diluted in MHB and dispensed into a 96-well round bottom microtiter plate (Costar) to a final concentration range of 128− 2 μg/mL. Aliquots of 0.5 McFarland standardized inoculum of bacteria were dispensed into wells containing polyamine compounds to a final inoculum of 5 × 10 5 CFU/mL. Bacterial cultures were grown statically at 37°C for 24 h (Thermo Scientific Heratherm). The MIC was defined as the lowest concentration of compound to result in no visible growth measured through inspection of turbidity. Tobramycin sulfate and kanamycin sulfate were used as antibiotic (positive) controls.

Ex Vivo Hemolytic Assay
Informed consent was gained from three healthy donors prior to blood donation. Experiments were approved by the University of Bath, Research Ethics Approval Committee for Health (REACH) [reference: EP 18/19 108]. Whole blood (10 mL) was obtained from three healthy donors, drawn directly into K 2 -EDTA-coated Vacutainer tubes (BD Biosciences) to prevent coagulation. Tubes were centrifuged at 500g for 10 min at 4°C (Eppendorf 5810R), the plasma layer was aspirated, discarded, and the remaining hematocrit was filled to the original volume with sterile saline solution (150 mM NaCl) and gently mixed by inversion. Blood cells were washed three times according to this above procedure, then after the final wash, hematocrit was resuspended in sterile phosphate buffered saline (PBS; Oxoid) to the original volume. Hematocrit was diluted to 2% (v/v) in PBS. Blood cells were aliquoted (100 μL) in triplicate in a 96-well microtiter plate. Equal volumes of polyamine compound diluted in PBS were added to a final concentration of 512−16 μg/mL and incubated for 1 h at 37°C. Blood cells incubated with saline served as a measure of spontaneous lysis of erythrocytes and therefore as a negative control buffer. Total (hemo)lysis of erythrocytes was obtained following incubation with Triton X-100 (Sigma-Aldrich, UK) (2% v/v) as a positive control. Following incubation, plates were centrifuged at 500g for 5 min and 100 μL of supernatant was transferred to a new 96-well plate and the absorbance (OD 404nm ) was measured using a Sunrise absorbance microplate reader (Tecan Life Sciences). Degree of hemolysis was expressed as % hemolysis (see equation) relative to spontaneous lysis controls = × % hemolysis (corrected absorbance of sample cells /correct absorbance of lysed cells) 100 Each experiment was performed using three technical repeats, and values were derived from three biological replicates from three different blood donors.

Instrumentation
NMR spectra including 1 H, 13 C, HSQC, HMBC, were recorded on Bruker Avance III (operating at 500.13 MHz for 1 H and 125.77 MHz for 13 C) spectrometers at 25°C. MestReNova has been used for processing the spectra. 1 H and 13 C chemical shifts (δ) were observed and are reported in parts per million (ppm) relative to tetramethylsilane at 0.00 ppm as an internal reference or to the residual solvent peak, HDO at 4.79 ppm. High resolution time-of flight mass spectra were obtained on a Bruker Daltonics "micrOTOF" mass spectrometer using electrospray ionization (ESI) (loop injection +ve ion mode). PerkinElmer 65 spectrum FT-IR spectroscopy was used to obtain the IR spectra. Anhydrous potassium bromide (KBr) discs were prepared for solid samples.

General Procedure A: Boc Removal
A solution of fully Boc polyamine in DCM (9 mL) was deprotected by adding TFA (1 mL) at 20°C. The solution was stirred for a further 18 h, then concentrated in vacuo and lyophilized to yield the desired product as a pale yellow oil (poly-TFA salt).